Fas (CD95/APO-1) and its specific ligand (FasL/CD95L) are members of the tumor necrosis factor (TNF) receptor and TNF families of proteins, respectively. (Nagata, S. et al. Science 267, 1449-1456 (1995). Interaction between Fas and FasL triggers a cascade of subcellular events that results in a definable cell death process in Fas-expressing targets. Fas is a 45 kDa type I membrane protein expressed constitutively in various tissues, including spleen, lymph nodes, liver, lung, kidney and ovary. (Leithauser, F. et al. Lab Invest 69, 415-429 (1993); Watanabe-Fukunaga, R. et al. J Immunol 148, 1274-1279 (1992)). FasL is a 40 kDa type II membrane protein, and its expression is predominantly restricted to lymphoid organs and perhaps certain immune-privileged tissues. (Suda, T. et al. Cell 75, 1169-1178 (1993); Suda, T. et al. J Immunol 154, 3806-3813 (1995)). In humans, FasL can induce cytolysis of Fas-expressing cells, either as a membrane-bound form or as a 17 kDa soluble form, which is released through metalloproteinase-mediated proteolytic shedding. (Kayagaki, N. et al. J Exp Med 182, 1777-1783 (1995); Mariani, S. M. et al. Eur J Immunol 25, 2303-2307 (1995)).
The FasL/Fas system has been implicated in the control of the immune response and inflammation, the response to infection, neoplasia, and death of parenchymal cells in several organs. (Nagata et al. supra; Biancone, L. et al. J Exp Med 186, 147-152 (1997); Krammer, P. H. Adv Immunol 71, 163-210 (1999); Seino, K. et al. J Immunol 161, 4484-4488 (1998)). Defects of the FasL/Fas system can limit lymphocyte apoptosis and lead to lymphoproliferation and autoimmunity. A role for FasL-Fas in the pathogenesis of rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, viral hepatitis, renal injury, inflammation, aging, graft rejection, HIV infection and a host of other diseases has been proposed. (Famularo, G., et al. Med Hypotheses 53, 50-62 (1999)). Fas mediated apoptosis is an important component of tissue specific organ damage, such as liver injury which has been shown to be induced through the engagement of the Fas-FasL receptor system. (Kakinuma, C. et al. Toxicol Pathol 27, 412-420 (1999); Famularo et al. supra; Martinez, O. M. et al. Int Rev Immunol 18, 527-546 (1999); Kataoka, Y. et al. Immunology 103, 310-318 (2001); Chung, C. S. et al. Surgery 130, 339-345 (2001); Doughty, L. et al. Pediatr Res 52, 922-927 (2002)). Consequently, the FasL-Fas pathway represents an important general target for therapeutic intervention.
Monoclonal anti-FasL antibody and recombinant soluble Fas protein are well recognized potential candidate antagonists for clinical studies. (Hashimoto, H. et al. Arthritis and Rheumatism 41, 657-662 (1998); Kanda, Y. et al. Bone Marrow Transplantation 22, 751-754 (1998); Kato, K. et al. British Journal of Haematology 103, 1164-1166 (1998); Maggi, C. A. Pharmacological Research 38, 1-34 (1998)). Attempts to neutralize FasL with antibodies has been examined in a variety of animal models. (Okuda, Y. et al. Biochem Biophys Res Commun 275, 164-168 (2000)). While antibodies have a long half life and are highly specific they also have important limitations: (i) commercial-scale production may be either difficult or costly, (ii) conformational stability may vary with the environment of the body fluids, (iii) antibodies may be excluded from certain compartments e.g., the brain, due to failure to cross the blood/brain barrier, and (iv) they may lead to the development of neutralizing antibodies, etc. (Cho, M. J. et al. Trends Biotechnol 14, 153-158 (1996)).
Many disadvantages of large macromolecules can be overcome by creating small molecular inhibitors that are targeted to surface receptors or their ligands. Peptidomimetics that are constructed to resemble secondary structural features of the targeted protein represent an approach to overcome some of the limitations of macromolecules and can mimic inhibitory features of large molecules such as antibody (Park, B. W. et al. Nat Biotechnol 18, 194-198 (2000)) and soluble receptors. (Takasaki, W. et al. Nat Biotechnol 15, 1266-1270 (1997)). Recently several peptidomimetics that inhibit ligand-receptor binding and that mediate potent biological effects have been described. (Park et al. supra; Takasaki, et al. supra). These peptides represent novel small molecular tools that can act with potency comparable to or equivalent to the natural antagonist. (Takasaki et al. supra; Wrighton, N. C. et al. Nat Biotechnol 15, 1261-1265 (1997)).
Several studies suggest that the presence of FasL in the eye is a barrier to both inflammatory cells (Griffith, T. S., et al., Science, 270, 1189-92 (1995); Gao, Y., et al., J Exp Med., 188, 887-96, (1998)) and development of new blood vessels (Kaplan, H. J., et al., Nat. Med., 5, 292-97 (1999)). The control of inflammation is known to be a component of the immune privilege of the eye (Griffith et al., 1995, supra; Griffith, T. S., et al., Immunity, 5, 7-16 (1996); Greil, R., A., et al., Leukemia & Lymphoma, 31, 477, (1998); Oconnell, J., et al., Molecular Medicine, 3, 294-300 (1997)). FasL expression in ocular tissue induces apoptosis in Fas+ lymphoid cells that invade the eye in response to viral infection or corneal grafting (Griffith, T. S., et al., 1995, supra; Stuart, P. M., et al., J Clin Invest, 99, 396-402 (1997); Chen, J. J., et al., Science, 282, 1714-1717 (1998); Mohan, R. R., et al., Experimental Eye Research, 65, 575-589 (1997)). FasL expression in the retina inhibits growth of blood vessels beneath the retina (Kaplan et al., supra), by inducing apoptosis in vascular endothelial cells that are known to express the Fas antigen. The loss of FasL expression in this region may be a predisposing factor in age related macular degeneration, allowing vessels to localize beneath the retina after penetration of Bruch's membrane. This process may lead to retinal detachment and visual loss.
FasL is also expressed in the cornea. Corneas that did not express functional FasL (gld) showed significantly greater neovascularization than normal corneas. In addition, engagement of Fas on vessels growing in vitro prevents vascular extension. These results suggest that FasL regulates neovascularization by engaging Fas on growing vessels and inducing apoptosis of the Fas+ vascular endothelial cells. Fas/FasL interaction is also required for the antiangiogenic effects of IL-12 and IL-2 when treating murine renal carcinoma (Wigginton, J. M., et al., J Clin Invest., 108, 51-62 (2001)).
Corneal neovascularization may be due to a complex interplay between several anti-angiogenic factors and Fas. This is evident from the fact that while gld and lpr mice are less prone to spontaneous neovascularization, normal development of eye is unaffected. This is similar to observations made with the immune privilege of the eye, where FasL works in concert with other inhibitory agents to control the spread of inflammation (Stuart, P. M., et al., Invest Ophthalmol Vis Sci., 44, 93-98 (2003)). The role of FasL seems to be critical when the eye is challenged or stimulated with an agent that induced inflammation (Griffith et al., 1995, supra; Kaplan et al., supra) or growth factors such FGF, HGF etc. (Stuart, et. al., supra).
Recently, an inhibitor responsible for the avascularity of ocular compartments was identified in the cornea as pigment epithelium-derived factor (PEDF) (Dawson, D. W., et al., Science, 285, 245-48 (1999)). This protein has been shown to have neurotrophic activity (Tombran-Tink, J., et al., J Neurosci, 15, 4992-5003 (1995); Taniwaki, T., et al., J Neurochem, 68, 26-32 (1997)) but is now known as a potent anti-angiogenic molecule (Gettins, P. G., et al., Biol Chem, 383, 1677-82 (2002); Simonovic, M., et al., Proc Natl Acad Sci USA, 98, 11131-35 (2001)). It seems to be a constitutive component of ocular compartments, and neutralization of its activity permits new vessel growth into the central cornea (Dawson et al., supra). Apoptosis in endothelial cells is associated with the activity of PEDF (Stellmach, V., et al., Proc Natl Acad Sci USA, 98, 2593-2597 (2001); Volpert, O. V., et al., Nat Med, 8, 349-357 (2002)). It has been shown that PEDF inhibitory function is orchestrated via upregulation of Fas (Volpert, O. V., et al., supra) in the cornea to inhibit spontaneous neovascularization and limit induction of angiogenesis.
The pattern of Fas L expression in the eye and the results obtained when FasL expression is modified in ocular tissue suggest that fodifying Fas function might be useful for various eye related pathologies. Altering Fas function in the eye may be a useful strategy, for example, for inhibiting corneal neovascularization.
The present inventors have undertaken a structural analysis Fas-FasL interactions. The inventors have developed an interaction model of FasL and Fas created by computer assisted modeling and designed peptide mimetics based on the deduced secondary structural features of Fas. The understanding of Fas-FasL recognition features at the atomic level has allowed the inventors to design mimetics that modulate Fas signaling functions and can thus be used to treat Fas pathologies.
Accordingly, the present inventors have identified and developed peptidomimetics that alter Fas function and will therefore have therapeutic applications against disease states mediated by Fas. The mimetics were effective in inhibiting β-FGF induced corneal neovascularization in vivo, demonstrating that Fas-FasL interaction plays a significant role in regulating extension of new blood vessels into the cornea, and indicating that Fas is a therapeutic target for eye related pathologies. The in vivo biological activity of the mimetics was also validated in a murine model of Fas-dependent Con A induced hepatitis injury. Accordingly, the mimetics are therefore also useful as therapy for fulminant hepatitis.